Equine lyme disease vaccines and methods

Abstract

A vaccine against Lyme disease is provided, comprising a B burgdorferi antigen and an adjuvant comprising oil, surfactant, and a polyoxyethylene-polyoxypropylene block copolymer. A method of using said vaccine to prevent Lyme disease in equines is also provided.

Description

Equine Lyme Disease Vaccines and MethodsFIELD OF THE INVENTION

[0001] This invention is generally in the field of equine vaccines against Lyme diseaseJOINT RESEARCH AGREENENT

[0002] Inventions disclosed herein have been made pursuant to and in scope of an existing Joint Research Agreement between Zoetis LLC and Virginia Commonwealth Intellectual Property Foundation drawn to development of veterinary vaccines against Lyme disease.SEQUENCE LISTING INFORMATION

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 23, 2024, is named ZP000399A.xml and is 54,415 bytes in size.BACKGROUND

[0004] Lyme disease is a bacterial infection caused by pathogenic spirochetes of the genus Borrelia. The infection can occur in humans, dogs, deer, horses, mice and other animals, and is transmitted by arthropod vectors, most notably ticks of the genus Ixodes. Borrelia burgdorferi (B burgdorferi), the most common cause of Lyme disease in North America, was first cultured in 1982. Borrelia are introduced into the host at the site of the tick bite and this is also the location of the initial characteristic skin lesion, erythema chronicum migrans (ECM).

[0005] The infection may be treated at any time with antibiotics such as doxycycline, penicillin, erythromycin, tetracycline, and ceftriaxone. Once infection has occurred, however, the drugs may not purge the host of the spirochete, but may only act to control the chronic forms of the disease. Complications such as arthritis and fatigue may continue for several years after diagnosis and treatment.

[0006] The canine Lyme disease vaccines were developed to provide protection by primarily inducing OspA borreliacidal antibodies. B. burgdorferi OspC is another potential target for borreliacidal antibody-mediated immunity. This protein appears to have an epitope that is responsible for inducing borreliacidal antibodies, and is not conserved among the pathogenicBorrelia spp. Although the specific function of the OspC protein remains unknown, it has been suggested that OspC expression is required for infection of mammals, but not for infection of ticks. Borrelia express OspC shortly after the tick begins feeding and must continue to express OspC in order to establish an infection in mammals. Therefore, the "window of effectiveness" of the OspC borreliacidal antibodies is increased significantly, compared to OspA borreliacidal antibodies.

[0007] Callister et al., (US Patents 6,210,676 and 6,464,985, incorporated by reference herein) have suggested employing an immunogenic polypeptide fragment of OspC, alone or in combination with an OspA polypeptide, to prepare a vaccine to protect humans and other mammals against Lyme disease. Livey et al. (U. S. Pat. No. 6,872,550, incorporated by reference herein) also proposed a vaccine for immunizing against Lyme disease prepared from a combination of recombinant OspA, OspB, and OspC proteins.

[0008] Lohse et al (US Patent 9,562,079, incorporated herein by reference) disclose a canine vaccine against Lyme disease containing antigens against OspA and OspC and adjuvanted with Alum.

[0009] Currently, there is no vaccine on the market that prevents Lyme disease in horses.SUMMARY OF INVENTION

[0010] In the first aspect, the instant disclosure provides a vaccine comprising a B burgdorferi antigen and an adjuvant, the adjuvant comprising a metabolizable oil, polyoxyethylenepolyoxypropylene block copolymer, optionally, a surfactant, and, optionally, a CpG-containing immunostimulatory oligonucleotide, wherein said vaccine is an oil-in-water (O / W) emulsion.

[0011] In a subset of embodiments, said B burgdorferi antigen comprises an OspA antigen and an OspC antigen. Preferably, said OspC antigen comprises a plurality of fragments from multiple OspC phylotypes, more preferably said OspC antigen comprises a chimeric protein comprising immunodominant epitopes of different OspC phylotypes. In the most preferred embodiments, said chimeric protein comprises SEQ ID NO: 40, or SEQ ID NO: 41 or a sequence that is at least 90% identical to SEQ ID NO: 40 or SEQ ID NO: 41. In a further subset of embodiments, said OspA antigen comprises SEQ ID NO: 43.

[0012] In certain embodiments, said CpG-containing immunostimulatory oligonucleotide a P-class immunostimulatory oligonucleotide characterized by the presence of one or more 5'-TLR-9 activating motif (s) and two palindromes or two complementarity areas. Preferably, said P-class immunostimulatory oligonucleotide is 5' modified. More preferably, said P class immunostimulatory oligonucleotide comprises at least 22 contiguous nucleotides of SEQ ID NO: 8. In certain embodiments, the CpG containing immunostimulatory oligonucleotide is present in the amount of about 20 to about 250 μg per dose.

[0013] In a further subset of embodiments, said metabolizable oil is squalane, and the optional surfactant is polyoxyethylene sorbitan monooleate.

[0014] In a particularly preferred set of embodiments, said metabolizable oil is present in the amount of about 0.2% to about 0.8% v / v of the vaccine; said polyoxyethylene-polyoxypropylene block copolymer is present in the amount of about 0.1% to about 0.4% v / v of the vaccine; and polyoxyethylene sorbitan monooleate is present in the amount of about 0.016% to about 0.064% v / v of the vaccine.

[0015] In a second aspect, the disclosure provides a method of protecting an equine in need thereof against Lyme disease, the method comprising administering to said equine the vaccine according to any one of the embodiments of the first aspect of the invention. In certain embodiments of this second aspect, the step of administering comprises a first administration of the vaccine according to any embodiment of the first aspect of the invention and a second administration of the vaccine according to any embodiment of the first aspect of the invention, wherein said first administration precedes said second administration by about 14 to about 42 days. In a subset of embodiments, the method further comprises a third administration of the vaccine according to any embodiment of the first aspect of the invention, wherein said third administration follows the second administration by about 14 to about 42 days.

[0016] In certain embodiments, said equine is about 3 months of age or older, preferably 6 months of age or older. Preferably, said equine has not been previously vaccinated against Lyme disease.

[0017] In certain embodiments, the method disclosed herein further comprises re-vaccination of said equine 18 months after the previous dose of the vaccine according to any embodiment of the first aspect of the invention.DETAILED DESCRIPTION

[0018] For a better understanding of the instant application, the following non-limiting definitions are provided:

[0019] The term "about" as applied to a reference number refers to the reference number plus or minus 10 percent of said value.

[0020] The term "at least 95% identical" includes all percentages of identity including and between 95% and 100%, for example, 96%, 97%, 98%, 99%, etc.

[0021] The term "alpha helix 5 region" or "helix 5 region" refers to amino acid sequence located between residues 160 and 200 of OspC phylotype A strain B31 and contains secondary structural elements including a portion of loop 6, alpha helix 5, and the unstructured C-terminal domain (Kumaran et aL, 2001).

[0022] The term "conservative substitution" denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue. Examples of conservative substitution include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue, or the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, and the like. The term "conservative substitution" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.

[0023] The term "conservative variation" of a reference protein or a reference nucleic acid refers to a protein or a nucleic acid, respectively, which differs from the reference molecule by only conservative substitution(s).

[0024] The term "construct" preceded by a phylotype name (e.g., N-construct or I-construct) refers to an amino acid sequence comprising the loop peptide and the helix peptide.

[0025] The term "helix peptide" or "alpha helix peptide" of a certain phylotype of OspC refers to a peptide which is at least 95% identical to an immunodominant epitope from alpha helix 5 region of OspC protein of that phylotype. Thus, for example, helix peptide N refers to a peptide which is at least 95% identical to an immunodominant epitope from alpha helix 5 region of OspC phylotype N.

[0026] The term "immunodominant epitope" refers to an epitope on a molecule that induces a dominant, or intense, immune response when compared to other epitopes, including one or both B- and T-cell responses.

[0027] The term "linear epitope" refers to an epitope comprising a single, non-interrupted, contiguous chain of amino acids joined together by peptide bonds to form a peptide or polypeptide. Such an epitope can be described by its primary structure, i.e. the linear sequence of amino acids in the chain. Such an epitope, when expressed in a recombinant protein subunit of OspC, retains the ability to bind infection-induced antibodies in a manner similar to the binding of wild-type protein.

[0028] The term "loop peptide" of a certain phylotype of OspC refers to a peptide which is at least 95% identical to an immunodominant epitope from loop 5 region of OspC protein of that phylotype. Thus, for example, loop peptide N refers to a peptide which is at least 95% identical to an immunodominant epitope from loop 5 region of OspC phylotype N. The term "loop 5 region" refers to amino acid sequence generally located between residues 131 and 159 of OspC phylotype A strain B31 and contains secondary structural elements, including a portion of alpha helix 3, loop 5 and alpha helix 4. See Kumaran et al., 2001. The sequence for OspC phylotype A strain B31 is provided in SEQ ID NO: 11.

[0029] The term "therapeutically effective amount" as used herein means an amount of a microorganism, or a subunit antigen, or polypeptides, or polynucleotide molecules, and combinations thereof, sufficient to elicit an immune response in the subject to which it is administered. The immune response can comprise, without limitation, induction of cellular and / or humoral immunity.

[0030] The terms "vaccine" and "vaccine composition," as used herein, mean a composition which prevents or reduces an infection, or which prevents or reduces one or more signs orsymptoms of infection. The protective effects of a vaccine composition against a pathogen are normally achieved by inducing in the subject an immune response, either a cell-mediated or a humoral immune response or a combination of both. Generally speaking, abolished or reduced incidences of infection, amelioration of the signs or symptoms, or accelerated elimination of the microorganism from the infected subjects are indicative of the protective effects of a vaccine composition.

[0031] In a broad aspect, the instant invention provides a vaccine for prevention of Lyme disease in equines. Broadly, the vaccine comprises a B burgdorferi antigen and an adjuvant, the adjuvant comprising a CpG containing immunostimulatory oligonucleotide, a metabolizable oil, a copolymer, and, optionally, a surfactant. Preferably, the vaccine is formulated as an oil-in-water emulsion.Antigen

[0032] Multiple B burgdorferi antigens are suitable for use in the vaccines described herein. In certain embodiments, the antigen comprises inactivated B burgdorferi. In other embodiments, the B burgdorferi antigen is recombinantly made. Preferably, it comprises an OspA antigen and an OspC antigen capable of inducing antibodies against OspA and OspC proteins, respectively, of B burgdorferi. Preferably, the OspC antigen comprises epitopes from different phylotypes.

[0033] Thus, in certain embodiments the vaccine may include two proteins: the first protein comprising an OspA or a fragment thereof, and a chimeric protein, comprising an OspC protein or a fragment thereof. In some embodiments, the chimeric protein comprises multiple fragments of OspC proteins of different phylotypes.

[0034] In some embodiments, the first protein comprises a fragment of OspA protein (SEQ ID NO: 12) (MGKQNVSSLDEKNSVSVDLPGEMNVLVSKEKNKDGKYDLIATVDKLELKGTS DKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRAD GTRLEYTEIKSDGSGKAKEVLKSYVLEGTLTAETTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWN SGTSTLTITVNSK KTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALK), which is immediately downstream of a viral protein, such as, for example, a fragment of the influenza virus NS1 protein, which is SEQ ID NO: 13 (MDPNTVSSFQVDSFLWHVRKRVADQELGDAPFLDRLRRDQKSLRGRGSTLG LDIETATRAGKQIVERILKEESDEALKMT). An important requirement for the first protein is its abilityto generate anti-OspA antibodies in a vaccinated animal. Thus, the full-length sequence of the OspA fragment is not necessary, and neither is the 100% identity to SEQ ID NO: 12.

[0035] As noted elsewhere in the application, 90% sequence identity is likely to be sufficient to provide suitable level of antibody production. The differing amino acids can be conservative substitutions, and / or are located outside of immunodominant epitope(s) of the OspA fragment.

[0036] In other embodiments, shorter OspA fragments can be used. A person of ordinary skill in the art would know how to determine which OspA fragments contain immunodominant epitopes capable of generating borreliacidal antibodies.

[0037] The inventors have surprisingly found that the first protein comprising, from N- to C-terminus, a fragment of the influenza virus NS-1 protein, followed by OspA protein with its signal sequence removed, is particularly suitable for the immunogenic compositions and vaccines of the instant invention.

[0038] Prior art studies are silent as to what phylotypes of OspC are prevalent in invasive Lyme disease in equines. Most studies have been performed on human samples. Jones et al reports that the most prevalent phylotypes found in joint fluid of the human patients with arthritis are K and A, and typically, and phylotypes A, B, C, D, H, K, N were discovered. Arthritis Rheum 2009 60(7) 2174. Earnhart et al have discovered phylotypes A, B, I, K, C, D, N in blood and / or CSF samples Infect Immun. 2005 73(12): 7869. Other studies typically associated phylotypes A, B, I and K with invasive forms of Lyme disease in humans.

[0039] According to some embodiments, the chimeric protein contains immunodominant epitopes capable of generating immune response against different OspC protein phylotypes. More specifically, the chimeric protein comprises immunodominant epitopes of multiple phylotypes of OspC. The immunodominant epitopes may be in the form of loop and / or helix peptides as discussed below, or they may be present within larger fragments of the target OspC protein. A suitable non-limiting example of such fragments is SEQ ID NO: 42 (NNSGKDGNTSANSADESVKGPNLTEISKKITESNAVVLAVKEIETLLSSIDELATKAIGQKIDANGLGVQANQ NGSLLAGAYAISTLITQKLSALNSEDLKEKVAKVKKCSEDFTNKLKNGNAQLGLAAATDDNAKAAILKTNGTN DKGAKELKDLSDSVESLV KAAQVMLTNSVKELTSPVVAESPKKP), which is a fragment of OspC phylotype F protein.

[0040] Previous studies demonstrate that the inclusion of the longerfragment of one of the OspC phylotypes (e.g., phylotype F) is beneficial for the expression level and thus makes the manufacturing of the chimeric protein more efficient.

[0041] Buckles et al demonstrated that loop 5 of OspC protein is surface exposed and may be a suitable target for generating borreliacidal antibodies. Clin Vaccine Immunol. 2006 October; 13(10):1162-5. See also WO09135118. However, considering that at least 21 phylotypes of OspC have been described (Seinost et aL, Infect Immun. 1999 July; 67(7):3518-24 1999), it remains to be determined what combination provides suitable protection against Lyme disease.

[0042] Thus, in some embodiments, the chimeric protein comprises linear epitopes from loop 5 region (loop peptides) and helix 5 regions (helix 5 peptides) of OspC proteins of different phylotypes. Currently considered phylotypes are T, U, E, A, B, K, I, H, N, C, M, D and F. The chimeric protein may thus comprise loop and helix peptides from 2-13 phylotypes of OspC, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 phylotypes. The order of the peptides is not crucial. In some embodiments, the loop peptides are interspaced with the helix peptides, and vice versa. In other words, the loop and helix peptides are arranged sequentially: in such embodiments, no two loop peptides should be present in the chimeric protein without a helix peptide between them, and no two helix peptides should be present without a loop peptide between them.

[0043] A person of ordinary skill in the art would be aware how to determine immunodominant epitopes from the loop regions and helix regions of various OspC phylotypes. For example, sera from subjects infected with B burgdorferi of different phylotypes may be reacted with specific peptides from the loop regions and helix regions of the corresponding phylotypes, and the binding of the antibodies present in the sera to the loop peptides and / or helix peptides can be quantified (e.g., by ELISA, immunoblot, etc), thus providing clues as to which peptides contain immunodominant linear epitopes from a given OspC phylotype.

[0044] Similarly, the borreliacidal activity of the antibodies may be determined by methods well known in the art, e.g., generally, by co-incubating cultured B burgdorferi with the sera from subjects challenged with the immunodominant linear epitopes as described above, and quantification of living and dead Borrelia.

[0045] In some embodiments, the sequences for the loop peptides and helix peptides are as follows:Loop peptide I is at least 95% identical to SEQ ID NO: 14 (AKLKGEHTDLGKEGVT);Helix peptide I is at least 95% identical to SEQ ID NO: 15 (KGADELEKLFESVKNLSKAAKEMLTNSVKE);Loop peptide H is at least 95% identical to SEQ ID NO: 16 (SEKFAGKLKNEHASLGKKDAT); Helix peptide H is at least 95% identical to SEQ ID NO: 17 (KGAKELKDLSDSVESLVKA);Loop peptide N is at least 95% identical to SEQ ID NO: 18 (SDDFTKKLQSSHAQLGVAGGATT); Helix peptide N is at least 95% identical to SEQ ID NO: 19 (ADELEKLFKSVESLAKAAQDALANSVNELTS);Loop peptide C is at least 95% identical to SEQ ID NO: 20 (KKLKEKHTDLGKKDAT);Helix peptide C is at least 95% identical to SEQ ID NO: 21 (AAELEKLFESVENLAKAAKEMLSNS); Loop peptide M is at least 95% identical to SEQ ID NO: 22 (NKAFTDKLKSSHAELGIANGAAT); Helix peptide M is at least 95% identical to SEQ ID NO: 23 (KGAQELEKLFESVKNLSKAAQETLNNSVKE);Loop peptide D is at least 95% identical to SEQ ID NO: 24 (SESFTKKLSDNQAELGIENAT);Helix peptide D is at least 95% identical to SEQ ID NO: 25 (KGAEELVKLSESVAGLLKAAQAILANSVKELTSPVVAESPKKP);Loop peptide F is at least 95% identical to SEQ ID NO: 26 (SEDFTNKLKNGNAQLGLAAAT); Helix peptide F is at least 95% identical to SEQ ID NO: 27 (KGAKELKDLSDSVESLVKAAQVMLTNS);Loop peptide T is at least 95% identical to SEQ ID NO: 28 (STGFTNKLKSGHAELGPVGGNAT); Helix peptide T is at least 95% identical to SEQ ID NO: 29 (KGAKELKDLSESVEALAKAAQAMLTNS);Loop peptide U is at least 95% identical to SEQ ID NO: 30 (SEKFTKKLSESHADIGIQAAT);Helix peptide U is at least 95% identical to SEQ ID NO: 31 (KGAEELDKLFKAVENLSK);Loop peptide E is at least 95% identical to SEQ ID NO: 32 (STEFTNKLKSEHAVLGLDNLT);Helix peptide E is at least 95% identical to SEQ ID NO: 33 (KGAAELEKLKAVENLSKAAQDTLKNAVKELTSPIVAESPKKP);Loop peptide A is at least 95% identical to SEQ ID NO: 34 (SETFTNKLKEKHTDLGKEGVT);Helix peptide A is at least 95% identical to SEQ ID NO: 35 (KGAEELGKLFESVEVLSKAAKEMLANSVKELTS);Loop peptide B is at least 95% identical to SEQ ID NO: 36 (SEEFSTKLKDNHAQLGIQGVT); Helix peptide B is at least 95% identical to SEQ ID NO: 37 (KGVEELEKLSGSLESLS);Loop peptide K is at least 95% identical to SEQ ID NO: 38 (SEDFTKKLEGEHAQLGIENVT); and Helix peptide K is at least 95% identical to SEQ ID NO: 39 (AAELEKLFKAVENLAKAAKEM).

[0046] In some embodiments, loop and helix peptides from the same phylotype are positioned together, i.e., adjacent to each other. For example, loop peptide from OspC phylotype A and a helix peptide from OspC phylotype A should not be separated by either the loop or the helix peptide from any other OspC phylotype.

[0047] Further, while in some embodiments, the loop and helix peptides from the same OspC phylotype are immediately adjacent to each other, in other embodiments, the loop peptide and the helix peptide may be separated by a linker sequence which does not affect the structure of the final protein. The properties of amino acids and their effects on protein structure are well known in the art and persons of ordinary skill in the art would be able to recognize which amino acids are suitable for the linkers.

[0048] While the order of the loop and helix peptides from different phylotypes is not crucial, in some embodiments, the chimeric protein comprises, in N- to C-orientation, an I-construct, a H-construct, a N-construct, a C-construct, a M-construct, a D-construct, followed by an amino acid sequence which is at least 90% identical to a fragment of OspC phylotype F protein (e.g., SEQ ID NO: 40). Thus, in some embodiments, the chimeric protein will comprise an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and preferably, 100%) identical to SEQ ID NO: 41.

[0049] In other embodiments, the loop and the helix peptides from phylotypes F, T, U, E, A, B, K are included within the chimeric protein. In some embodiments, the chimeric protein, thus, would comprise the following, in N- to C-orientation: a T-construct, a U-construct, a E-construct, an A-construct, a B-construct, a K-construct, the I-construct, the H-construct, the N-construct,the C-construct, the M-construct, and the D-construct. Optionally, the chimeric protein can also comprise an F-construct, which is, in some embodiments, is upstream of the T-construct. Alternatively, or additionally, the chimeric protein can contain the amino acid sequence which is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and preferably, 100%) identical to the fragment of OspC phylotype F protein.

[0050] Other suitable examples of the chimeric protein, as well as methods of making and using same are provided in Application PCT / US2011 / 056854 (filed on Oct. 19, 2011, inventors R. Marconi and C. Earnhart).

[0051] In certain embodiments, the vaccine would comprise SEQ ID NO: 43; and either one of SEQ ID NO: 40 or SEQ ID NO: 41. In the most preferred embodiment, the vaccine comprises SEQ ID NO: 43 and SEQ ID NO: 45.

[0052] As described above, the sequences present in the antigens do not need to be 100% identical to the reference sequences. Identity of 90% or more (and preferably 95% or more, or 96% or more, or 97% or more or 98% or more, or 99% or more) is likely to be effective in eliciting similar immune response.

[0053] The skilled person will further acknowledge that alterations of the nucleic acid sequence resulting in modifications of the amino acid sequence of the protein it codes may have little, if any, effect on the resulting three-dimensional structure of the protein. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, ora more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in the substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a protein with substantially the same functional activity.

[0054] Preferably, the differences from the reference sequences are substitutions. At least some of these constitutions (e.g., at least 50%, or at least 60% or at least 70% or at least 80% or at least 90% or at least 95% or 100%) are conservative substitutions. Conservative substitutions are known in the art. Previous research identified several groups of conservative substituents. The following six groups each contain amino acids that are typical conservative substitutions for oneanother: [1] Alanine (A), Serine (S), Threonine (T); [2] Aspartic acid (D), Glutamic acid (E); [3] Asparagine (N), Glutamine (Q); [4] Arginine (R), Lysine (K), Histidine (H); [5] Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and [6] Phenylalanine (F), Tyrosine (Y), Tryptophan (W), (see, e.g., US Patent Publication 20100291549).

[0055] Protein and / or nucleic acid sequence identities can be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. For sequence comparison, typically one sequence acts as a reference sequence (e.g., a sequence disclosed herein), to which test sequences are compared. A sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0056] The sequences described herein may be manufactured by methods well known in the art. The polypeptides may be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield J. (1963) J Am Chem Soc 85:2149-2154). Peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.), in accordance with the instructions provided by the manufacturer. For example, subsequences may be chemically synthesized separately and combined using chemical methods to provide full-length polypeptides or fragments thereof. Alternatively, such sequences may be ordered from any number of companies which specialize in production of polypeptides. Most commonly, polypeptides may be produced by expressing coding nucleic acids and recovering polypeptides, as described below.

[0057] For example, in embodiments where loop peptides and the helix peptides are 100% identical to the fragments of OspC proteins of the target phylotypes, the nucleic acid sequences of such loop and helix peptides are also known or easily accessible from publicly available databases, e.g., Genbank. If the selected loop / helix peptides are somewhat different from the naturally occurring fragments of OspC proteins, the encoding nucleic acid sequencescan be easily designed using well known genetic code.

[0058] Many organisms display bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference or codon bias, differences in codon usage between organisms, is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, since the majority of amino acids are encoded by multiple codons (methionine is the exception), the nucleic acid sequences can be tailored for optimal gene expression in a given organism based on codon optimization.

[0059] Methods for producing recombinant polypeptides are also included. One such method comprises introducing into a population of cells any nucleic acid as described above, which is operatively linked to a regulatory sequence effective to produce the encoded polypeptide, culturing the host cells (e.g., yeast, insect, mammalian cells, plant cells, etc) in a culture medium to express the polypeptide, and isolating the polypeptide from the cells or from the culture medium. The nucleic acid is introduced into such cells by any delivery method as is known in the art, including, e.g., transformation, transfection, injection, gene gun, passive uptake, etc. As one skilled in the art will recognize, the nucleic acid may be part of a vector, such as a recombinant expression vector, including a DNA plasmid vector, or any vector as known in the art.

[0060] Alternatively, cell-free prokaryotic or eukaryotic-based expression systems may be used.

[0061] In some embodiments, the nucleic acid sequence encoding the first and / or the chimeric protein, may further comprise a sequence encoding a polypeptide (the "fusion partner") that is fused to the first and / or the chimeric protein, thereby facilitating purification of the fusion protein. In certain embodiments of this aspect of the invention, the fusion partner is a hexahistidine peptide (SEQ ID NO: 44, HHHHHH), as provided in the pQE vector (Qiagen, Inc.), and described in Gentz et al., Proc Natl Acad Sci USA 86:821-824 (1989), or it may be the HA tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell 37:767, 1984). The polynucleotide may also contain non-coding 5' and 3' sequences, suchas transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.

[0062] Adjuvant

[0063] The vaccine of the invention is adjuvanted with a CpG-containing immunostimulatory oligonucleotide, which is an oligodeoxynucleotide containing at least one unmethylated CG bond, a metabolizable oil, a polyoxyethylene-polyoxypropylene block copolymer, and, optionally, a surfactant, wherein said vaccine is formulated as an oil-in-water (O / W) emulsion.

[0064] Immunomodulatory oligonucleotides according to the invention comprise CpG (and are also referred to as "CpG containing immunostimulatory oligonucleotides", "CpG oligonucleotides" or simply "CpGs"). The effect of CpG containing oligonucleotides on the immune system has been known for over 20 years.

[0065] Generally, the CpGs suitable for the invention are between 8 and 100 bases long, e.g., between 10 and 50 bases long, or between 18 and 40 bases long or between 20 and 30 bases long, or 20-24 bases long.

[0066] Several classes of CpG have been described, including A-class CpGs, B-class CpGs, C-class CpGs, and P-class CpGs. In certain embodiments, the CpG containing immunostimulatory oligonucleotide is a P-class CpG. P-class CpGs are characterized by the presence of one or more TLR-9 activating motif(s) and two palindromes or two complementarity areas. Preferably, the one or more TLR-9 activating motifs are at the 5' of the oligonucleotide and may be completely or partially be incorporated into the 5' palindrome or the 5' complementarity area. TLR-9 activating motifs are known and include, without limitations, TCG, TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT, or TTTT. The 5' palindrome or the 5' complementary area is at least 6 bases long. The 3' palindrome or the 3' complementary area is at least 8 bases long and is generally rich in C and G. These structural features of the P-class CpGs confer the ability to spontaneously self-assemble into concatamers either in vitro and / or in vivo.

[0067] In order to increase lipophilicity of the CpG oligonucleotides, at least one lipophilic substituted nucleotide analog may be included, preferably at the 5' end of the oligonucleotide. The P-class immunostimulatory oligonucleotides may be modified according to techniques known in the art. For example, J-modification refers to iodo-modified nucleotides. E-modification refers to ethyl-modified nucleotide(s). Thus, E-modified P-class immunostimulatory oligonucleotides are P-class immunostimulatory oligonucleotides, wherein at least one nucleotide (preferably 5' nucleotide) is ethylated. Additional modifications include attachment of 6-nitro-benzimidazol, O-Methylation, modification with proynyl-dU, inosine modification, 2-bromovinyl attachment (preferably to uridine).

[0068] The oligonucleotides modified by an addition of a lipophilic moiety are generally described in US 20100166780.

[0069] In certain embodiments, CpGs according to the invention comprise the modified backbone including, without limitations, phosphorothioate modifications, halogenations, alkylation (e.g., ethyl- or methyl-modifications), and phosphodiester modifications. In certain embodiments, both modified and unmodified bonds are present in the CpG oligonucleotide according to the invention.

[0070] Suitable non-limiting examples of modified P-class immunostimulatory oligonucleotides are provided below ("*" refers to a phosphorothioate bond, refers to a phosphodiester bond, "JU" refers to 5'-lodo-2'-deoxyuridine and "EU" refers to 5-Ethyl-2'-deoxyuridine).SEQ ID NO: 1 5' T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G 3'SEQ ID NO: 2 5' T*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' SEQ ID NO: 3 5' T*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3' SEQ ID NO: 4 5' JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' SEQ ID NO: 5 5' JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C* G*T 3' SEQ ID NO: 6 5' JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C* G*T 3' SEQ ID NO: 7 5' EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' SEQ ID NO: 8 5' JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C* G*T 3' SEQ ID NO: 9 5' JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C* G*T3' SEQ ID NO: 10 5' T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G 3'

[0071] In certain embodiments, the CpG oligonucleotide according to the invention comprises any one of SEQ ID NOs 1-10 or an oligonucleotide comprising at least 15 consecutive bases of any one of SEQ ID NOs 1-10. In the most preferred embodiment, the vaccine comprises an oligonucleotide comprising at least 15 consecutive bases of SEQ ID NO: 8 (e.g., at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23).

[0072] The CpG oligonucleotide may be present in the vaccine in the amount of 10-400 |ig per dose of the vaccine, or 25-300 or 50-200 or 50-150 or about 100 |ig per dose.

[0073] In addition to the CpG-containing immunostimulatory oligonucleotide, the adjuvant comprises the metabolizable oil, the polyoxyethylene-polyoxypropylene block copolymer, and the surfactant.

[0074] Multiple metabolizable oils are known in the art including without limitation squalane, squalene, medium chain triglycerides, and long chain triglycerides. In one embodiment, the metabolizable oil is squalane. The metabolizable oil can be present in the vaccine in the amount of about 0.05% to about 35% v / v, e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, 1%, about 2%, about 3%, about 5%, about 8%, about 10%, about 15%, about 15%, about 20%, about 25%, or about 30% v / v. In certain preferred embodiments, the metabolizable oil is present in the amount of 0.1% to about 1% v / v, e.g., 0.3% to about 0.7%, or about 0.4% to about 0.5% v / v.

[0075] Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) triblock copolymers also known as poloxamers are also known in the art. They are composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). In certain embodiments, poloxamers are commercially available under the trade name PLURONIC®. The poloxamer can be present in the vaccine in the amount of amount of about 0.05% to about 0.8 % v / v.

[0076] The preferred Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) triblock copolymer is poloxamer which is commercially available under the trade names PLURONIC® L-121 and ETHOX® L-121 and has the formula of (C3H6O·C2H4O)x.

[0077] Polyoxyethylene-polyoxypropylene block copolymers are known to have surface active properties. Nevertheless, in certain preferred embodiments, the adjuvant may further optionally comprise an additional optional surfactant (sometimes referred to as a second surfactant or the optional second surfactant) that is not a polyoxyethylene-polyoxypropylene block copolymer. Multiple suitable surfactants are known in the art. Preferably, second surfactant, is an amphipathic, nonionic surfactant that is derived from ethoxylated sorbitan. In certain embodiments, the second surfactant is polyoxyethylene sorbitan monooleate also known asTWEEN® 80 or polysorbate 80. The optional surfactant can be present in the vaccine in the amount of amount of about 0.005% to about 10% v / v, e.g., about 0.01%, or about 0.02%, or about 0.03%, or about 0.04%, or about 0.05%, or about 0.06%, or about 0.07%, or about 0.08%, or about 0.09%, or about 0.1%, or about 0.15%, or about 0.20%, or about 0.5%, or about 0.8%, or about 1%, or about 5%, or about 8%, or about 9% v / v. In certain embodiments, the second surfactant us present in the amount from about 0.005% to about 0.1% v / v, or from about 0.01 to about 0.05% or from about 0.025% to about 0.04% v / v, or from about 0.1 % to about 0.5% v / v or from 0.25% to about 0.75% v / v.

[0078] A mixture of squalane, poloxamer 401, polyoxyethylene sorbitan monooleate and a buffered salt solution is known in the art and has been called "SP oil". In general, the SP oil emulsion contains about 1 to 3% vol / vol of polyoxyethylene-polyoxypropylene block copolymer, about 2 to 6% vol / vol of squalane, more particularly about 3 to 6% of squalane, and about 0.1 to 0.5% vol / vol of polyoxyethylene sorbitan monooleate, with the remainder being a buffered salt solution.

[0079] In certain embodiments, about 5% to about 20% SP oil is used in the vaccine. Thus, in certain embodiments, said metabolizable oil is present in the amount of about 0.2% to about 0.8% v / v of the vaccine; said polyoxyethylene-polyoxypropylene block copolymer is present in the amount of about 0.1% to about 0.4% v / v of the vaccine; and said polyoxyethylene sorbitan monooleate is present in the amount of about 0.016% to about 0.064% v / v of the vaccine.

[0080] In more specific embodiments, the metabolizable oil is present in the amount of about 0.4% v / v of the vaccine; said polyoxyethylene-polyoxypropylene block copolymer is present in the amount of about 0.2% v / v of the vaccine; and said polyoxyethylene sorbitan monooleate is present in the amount of about 0.032% v / v of the vaccine.

[0081] Vaccines described herein can further comprise pharmaceutically acceptable carriers, excipients and / or stabilizers (see e.g. Remington: The Science and practice of Pharmacy (2005) Lippincott Williams), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations, and may comprise buffers such as phosphate buffer with added 135-165 mM (e.g. about 150 mM) sodium chloride, succinate buffer (preferably, 10-40 mM succinate, more preferably 15-25mM sodium succinate, most preferably about 20 mM succinate), citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as Mercury((o-carboxyphenyl)thio)ethyl sodium salt (THIOMERSAL®), octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes, and other inactive ingredients. Ultimately, desired route of administration of the vaccines described herein would guide the choice of the inactive ingredients.

[0082] The vaccines described herein can be administered by any means that achieve the intended purpose. For example, route of administration of such a composition can be by parenteral, oral, oronasal, intranasal, intratracheal, topical, subcutaneous, intramuscular, transcutaneous, intradermal, intraperitoneal, intraocular, and intravenous administration. A combination of routes may also be used, including, without limitations, a first administration administered by injection (e.g., intradermally, subdermally, intramuscularly) and a second administration administered mucosally (e.g., orally or nasally or rectally or vaginally).

[0083] In one embodiment of the present invention, the composition is administered by intramuscularly. Parenteral administration can be by bolus injection or by gradual perfusion over time. Any suitable device may be used to administer the compositions, including syringes, droppers, needleless injection devices, patches, and the like. The route and device selected for use will depend on the composition of the adjuvant, the antigen, and the subject, and such are well known to the skilled artisan.

[0084] In certain embodiments, the vaccines are administered to the equines in need thereof in two administrations. The first administration precedes the second administration by about fourteen to about 42 (e.g., the second administration may follow the first administration by 15days, 21 days, 28 days, 35 days, or 40 days). Preferably, after the second administration, the vaccine exerts protection for at least six, or at least nine, or, more preferably, at least twelve months.

[0085] Optionally, the treatment regimen further comprises administering a third dose of the vaccine, said optional third dose preceding the second dose by about fourteen to about 42 (e.g., the third administration may follow the second administration by 15 days, 21 days, 28 days, 35 days, or 40 days). Preferably, after the second administration, the vaccine exerts protection for at least twelve, or at least 15, or, more preferably, at least 18 months.

[0086] Equines that receive the vaccines are foals, yearling, filly, colts, mares, stallions, geldings and can be equines of any sex and about 6 months of age or older.

[0087] All publications cited in the specification, both patent publications and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein fully incorporated by reference, to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

[0088] For a clearer understanding of the invention, the following examples are set forth below. These examples are merely illustrative, and are not understood to limit the scope or underlying principles of the invention in anyway. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the examples set forth hereinbelow and the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.EXAMPLESExample 1. Serum of horses vaccinated with Lyme disease antigen adjuvanted with SP oil / CpG combination exhibit Borreliacidal properties

[0089] Horses of mixed breed, male and female 32 horses (13 males and 19 females) approximately 1 year of age (11 to 14 months) were used for the study.

[0090] Inclusion Criteria: Horses were healthy and seronegative for B. burgdorferi antibodies which demonstrated animals had not been infected with or vaccinated for 8. burgdorferi prior to first vaccination.

[0091] Exclusion Criteria: No animals were excluded from the study. All horses were group housed in a single pen and pasture.

[0092] All facilities met or exceeded the requirements set forth in the current "Guide for the Care and Use of Agricultural Animals"

[0093] Horses were treated with a topical insecticide every two weeks per veterinary instructions throughout the study to reduce the likelihood that they would be naturally infected with B. burgdorferi.

[0094] Hay (or equivalent substitute) and grain was fed daily throughout the study. Water was available ad libitum. Animals were already acclimated to the housing facilities prior to study activities taking place.

[0095] The experimental design is summarized in Table 1:Table 1

[0096] In groups T04 and T05, SP Oil was used at 10% v / v. Thus, in the composition squalane is present in the amount of about 0.4% v / v of the vaccine, polyoxyethylene-polyoxypropylene block copolymer is present in the amount of about 0.2% v / v of the vaccine, and polyoxyethylene sorbitan monooleate is present in the amount of about 0.032% v / v of the vaccine. 1 MID of thevaccine contained 20 micrograms / dose of OspA antigen (SEQ ID NO: 43) and 30 micrograms / dose of OspC antigen (SEQ ID NO: 45, that comprises SEQ ID NO: 41 and N-terminal His-tag).

[0097] Injection site volumes (n / 6 x length x height x width) was calculated and listed by animal, treatment and time point. Frequency distributions of injection site swelling were calculated for each treatment and time point. Frequency distributions of ever having an injection site swelling were calculated for each vaccination as well as over all the vaccination periods.

[0098] Descriptive statistics including means, standard deviations, minimums and maximums were calculated for each treatment and time point. It was determined if an animal had a fever (> 102.5°F) at each time point and vaccination. Frequency distributions of fever were calculated for each time point and vaccination as well as overall.

[0099] Antibody titers were logarithmically transformed prior to analysis. The transformed titers were analyzed with a general linear mixed model for repeated measures. The model will include the fixed effects of treatment, time point and, treatment by time point interaction. The random effects in the model will be animal within treatment and residual. All possible pairwise treatment comparisons will be made at each time point using contrasts if the treatment main effect and / or the treatment by time point interaction is significant. Treatment least squares means at each time point, standard errors and 90% confidence intervals were back transformed to obtain the geometric means, back-transformed standard errors and their back- transformed confidence intervals if appropriate. In addition, minimums and maximums were calculated for each treatment and time point.

[0100] For the bactericidal assay setup, the viable bacteria number was determined by visual counting using wet-mount dark-field microscopy (400x magnification) after incubation with the relevant horse serum and GPS. Spirochetes must display both rotational (rotation around their axis) and translational (forward or reverse motion) motility. The total number of live bacteria was counted (5 fields of view, in triplicate tubes) for each sample and compared to the total number of live bacteria (5 fields of view, in triplicate tubes) in the negative control (NC). The number calculated as below was reported as percent killing.% Killinga= 100 - (# Live (sample)x 100)' # Live (NC) '[OOlOlJThe percent killing was analyzed with a general linear mixed model for repeated measurements. The fixed effects in the model was treatment, time point and treatment by time point interaction. The random effects in the model was animal within treatment and residual. Treatment least squares means, standard errors, 95% confidence limits, minimums and maximums were calculated for each time point. Contrasts was used to compare treatments at each time point.

[0102] Following each vaccination, all animals were normal with the exception of one animal in T03 that had signs of colic following the second vaccination. This animal was treated and returned to normal within the hour.

[0103] Only one injection site was observed with swelling: T05 caused some minor swelling lasting from one to two days after the second vaccination with no pain on palpation. The swelling resolved by the third day. No recurrence of swelling after the third vaccination. No other horses on the study experienced injection site observations. Injection site reactions are summarized in Table 2.Table 2. Frequency Distribution - Ever Having an Injection Site SwellingT01=B. burgdorferi vaccine (IxMID) with 1% REHYDRAGEL™T02=B. burgdorferi vaccine (IxMID) with 5.5% REHYDRAGEL ™ T03=B. burgdorferi vaccine (5xMID) with 1% REHYDRAGEL ™ T0^=B.burgdorferi vaccine (IxMID) with SP OilT05=B. burgdorferi vaccine (IxMID) with SP Oil+CpG

[0104] No fevers (temperatures > 102.5°C) were detected in any animals at any timepoint while on study.

[0105] Osp A & Osp C Antibody Titers.

[0106] Osp A and Osp C, vaccine components, are essential to the efficacy of the vaccine. Osp A antibodies are able to neutralize the bacterium residing within the tick. Osp C antibodies are neutralizing after infection. However, the antibody titer required to be considered "protective" is unknown.

[0107] The rapid increase in Osp A serum antibody titer after the first vaccination was significantly greater in the 5.5% REHYDRAGEL™ (T02) and the SP Oil+CpG (T05) groups compared to the commercial vaccine group (T01). Following the third dose, only the SP Oil+CpG (T05) group titer was significantly higher (Table 4).

[0108] The Osp C serum antibody titers were also significantly higher in the 5.5% REHYDRAGEL™ (T02) and SP Oil+CpG (T05) groups after the first dose. And after the third dose, all reformulated vaccine groups (T02 through T05) had significantly higher titers than the commercial vaccine group (T01) (Table 5).Table 1. Osp A Antibody Titers Least Squares MeansAsterisks indicate values significant at 0.10 level when compared to T01.Table 2. Osp C Antibody Titers Least Squares MeansAsterisks indicate values significant at 0.10 level when compared to T01.

[0109] Borrelia burgdorferi has a 39 kDa VIsE (VMP-like sequence E lipoprotein) containing the C6 region, so if an animal has antibodies to this region, it's indicative of Borrelia burgdorferi-caused disease, at least in other mammalian models such as the human and canine models. Accordingly, C6 antibodies are generated during an active infection and can correlate to infection load. All animals remained seronegative (<400) for C6 antibodies throughout the study, and therefore were not infected.[OOllOJOsp F antibodies are considered an indicator of a chronic infection of B. burgdorferi. A subset of the individual horses with low Osp F titers is shown in the table below. These low titers remained the same (±1 dilution) during the study indicating no response to infection and these low-level titers had no impact on the study outcome (<400 is considered negative)Table 6

[0111] Bactericidal Assay

[0112] The antibodies generated by vaccination mediate the killing of the bacteria through a complement dependent pathway. Combining a constant amount of exogenous complement with inactivated serum indicates the amount of functional antibody capable of inducing this pathway shown by the percent of bacteria killed in the assay.

[0113] There was a significant difference in the percent of bacteria killed from the SP Oil+CpG (T05) vaccinated group contrasted against all other treatments. Some increase in the percent of bactericidal activity was also seen in the 5.5% REHYDRAGEL™ (T02) and SP Oil (T04) groups against the control (T01) group, though not significant. Almost no difference was seen in the 5xMID (T03) group from the control (T01) group.Table 7. Least Squares Means of Percent Borrelia killed using Serum from Day 63Asterisks indicate values significant at 0.05 level when compared to T01.Table 8. P-values of Treatment Comparisons for 0.32% Serum testTable 9. P-values of Treatment Comparisons for 0.064% Serum test

[0114] The 1.6% serum dilution was saturated for all the groups (i.e. close to 100%). The largest range in percent killing among the treatment groups was seen using 0.064% serum in the assay, though the T05 group was still near the saturation level. The 1.6% serum test used a group pooled sample and provided a single result; while the other dilutions were tested individually and the results summarized. As the test serum dilution moved away from saturation in the assay, variation in results became apparent.Table 10. Result Variation for 0.32% Serum testTable 3. Result variation for 0.064% Serum testSummary and conclusions

[0115] Study Validity

[0116] All animals were healthy and seronegative for B. burgdorferi (Bb) antibodies prior to vaccination. All horses were seronegative (< 400) for C6, Osp A, and Osp C antibodies. The two horses with low serum antibodies titers (800) to Osp F prior to vaccination and continued on study. While an Osp F titer can indicate a chronic B burgdorferi infection, there is some assay variation tolerance to take into account. Antibody titers are not considered relevant until they are > 400, however, the assay tolerance is ± one dilution; in this case a 2-fold dilution. Therefore, an antibody titer of 800 is very low. This low titer coupled with the negative C6 titers on Day 0, and the continued Ce seronegative status at the study conclusion (Day 63) would indicate that the animals were not infected. One horse maintained the low 800 titer on Day 0 and Day 63. The other horse with the low 800 titer on Day 0 tested as seronegative (400) on Day 63. And one horse that was seronegative (400) on Day 0, tested with a low 800 titer on Day 63. All these titers are stable within the variation tolerance of the assay.

[0117] The study was considered valid even though it only partially met the criteria for inclusion since there were 2 horses that were > 400 for Osp F on Day 0.

[0118] Serologic Response

[0119] By the end of the study, the SP Oil+CpG (T05) vaccine response was significantly higher than the commercial (T01) vaccine response in all tests: Osp A titer 8 times higher, Osp C titer 20 times higher.

[0120] The SP Oil (T04) and 5.5 % Rehydragel™ (T02) vaccine responses were similar in all tests and slightly higherthan the commercial vaccine response: Osp A titers 2 times higher, Osp C titers 4 times higher.

[0121] The effect of the third dose was most notable in the Osp A antibody response. The Osp C antibody continued to increase after the third dose, but increased more sharply after the second dose for the SP Oil containing vaccines (T04 and T05).Example 2: Evaluation of the Serological Response after Administration of Experimental Borrelia Burgdorferi Bacterial Extract Vaccines in Horses

[0122] Eleven-to-fourteen-month-old male (castrated) and female mixed-breed horses were used in the study. The animals were seronegative to B. burgdorferi OspA, OspC and C6 tested by an ELISA assay (titer of 400 or less is considered seronegative). All horses were housed in two pens (pasture) with 16 animals per pen. All treatment groups were co-mingled in each pen. All facilities met or exceeded the requirements set forth in the current "Guide for the Care and Use of Agricultural Animals."

[0123] After randomization, horses were treated with a topical insecticide (EQUI-SPOT® or alternative) as per veterinary instructions throughout the study to reduce the likelihood of infection with B. burgdorferi through a natural infestation due to being housed outdoors.

[0124] Hay (or equivalent substitute) and grain were fed daily throughout the study. Water was available ad libitum. Animals had at least 7 days of acclimation to the new housing facilities prior to study activities taking place. Animals were randomly allocated to treatment groups using a generalized block design (8 per treatment group as above) using a SAS (SAS release 9.4 or higher, SAS Institute, Cary, NC) program developed specifically for the study with the ranuni function used to generate random numbers.

[0125] The experimental setup is provided in Table 12.Table 12

[0126] Blood (1 x 10 mL SST) was collected via jugular venipuncture on Days 0, 21, 42, (prior to vaccination) and 62. The samples were labeled with a unique sample id and processed to serum. SST blood samples were collected and processed at ambient temperatures. After processing, serum was divided into 2 or 3 aliquots. The aliquots were shipped to appropriate location and stored frozen at <-10° C until further testing.

[0127] C6 ELISA. Serum was tested on Days 0 and on the final day of the study (Day 63) using the C6 ELISA test to confirm animals were not exposed to B. burgdorferi prior to or during the study. Test results (quantitative: titers) was documented for B. burgdorferi (C6). Serum was tested for antibody levels (quantitative) to C6 by an ELISA. Streptavidin-coated plates were washed three times with PBS-Tween, approximately 300 pL per well, prior to the addition of antigen. After washing, the plates were coated with 100 pL of a final concentration of 25 ng / mL of biotinylated antigen per well in PBST and incubated for 60 ± 5 minutes at room temperature, shaking. Positive controls, negative controls and test serum samples were diluted in PBS-Tween to the appropriate start dilution. After transferring the test samples and control samples to the test plate, plates were incubated at room temperature for 60 + 5 minutes, shaking. Plates were washed three times with PBS-Tween, approximately 300 pL per well. Conjugate was diluted to the appropriate dilution in PBS-Tween and added at 100 pL per well. Plates were incubated at room temperature for 60 ± 5 minutes shaking, and then washed three times with PBS-Tween, approximately 300 pL per well. ABTS substrate was prepared and added to each well at 100 pL.Plates were incubated for 10-15 minutes at room temperature. Optical densities (OD) were determined, and test sample titers were calculated from the average plus three standard deviations of the negative control OD value. Test sample dilutions above the negative control OD represent the end point titer.

[0128] OspA and OspC ELISA. Serum was tested for antibody levels (quantitative) to OspA, OspC by an ELISA on all days of blood collection (Days 0, 21, 42, 63). Briefly, plates were coated with 100 pL of a final concentration of 2.5 pg / mL of coating antigen (OspA, OspC) per well in 0.01M Borate buffer. Plates were incubated overnight at 4°C. After discarding the coating solution, plates were blocked with 200 pL 1% Casein in PBS-Tween and incubated at 37°C for 60 ± 5 minutes. Blocked plates were washed three times with PBS-Tween, approximately 300 pL per well. Test serum samples were diluted in PBS-Tween to the appropriate start dilution. Positive and negative control samples were diluted accordingly for OspA, OspC. After transferring the test samples and control samples to the test plate, plates were incubated at 37°C for 60 ±5 minutes. Plates were washed three times with PBS-Tween, approximately 300 pL per well. Conjugate was diluted to the appropriate dilution in PBS-Tween and added at 100 pL per well. Plates were incubated at 37°C for 60 ±5 minutes and then washed three times with PBS-Tween, approximately 300 pL per well. ABTS substrate was prepared and added to each well at 100 pL. Plates were incubated for 10-15 minutes at room temperature. Optical densities (OD) were determined and test sample titers were calculated from the average plus three standard deviations of the negative control OD value. Test sample dilutions above the negative control OD represent the end point titer.Results

[0129] Median ELISA titers for OspC and OspA on days 20, 42, and 62 are provided in Tables 13 and 14, respectively.Table 13. Mean OspC ELISA titers on Study Day 20, 42, and 62Table 14. Mean OspA ELISA titers on Study Day 20, 42, and 62

[0130] These data demonstrate that all formulations elicit positive ELISA OspA and OspA titers. The formulations containing 100 micrograms CpG per dose and formulated with 0.063% phosphate buffer with 150 mM sodium chloride or with succinate buffer showed better titers than formulations buffered with 0.063% Phosphate buffer. The composition containing 20 mM succinate buffer elicited the greatest titer.

Claims

CLAIMS1. A vaccine comprising a B burgdorferi antigen and an adjuvant, the adjuvant comprising a metabolizable oil, polyoxyethylene-polyoxypropylene block copolymer, optionally, a surfactant, and, optionally, a CpG-containing immunostimulatory oligonucleotide, wherein said vaccine is an oil-in-water (O / W) emulsion.

2. The vaccine according to claim 1, wherein B burgdorferi antigen comprises an OspA antigen and an OspC antigen.

3. The vaccine of claim 2, wherein said OspC antigen comprises a plurality of fragments from multiple OspC phylotypes.

4. The vaccine of claim 3 wherein said OspC antigen comprises a chimeric protein comprising immunodominant epitopes of different OspC phylotypes.

5. The vaccine of claim 4, wherein said chimeric protein comprises SEQ ID NO: 40, or SEQ ID NO: 41 or a sequence that is at least 90% identical to SEQ ID NO: 40 or SEQ ID NO:

41.

6. The vaccine of any one of claims 1-5, wherein said OspA antigen comprises SEQ ID NO:43.

7. The vaccine of any one of claims 1-7, wherein said OspA antigen is present in the amount of about 20 micrograms per dose and wherein said chimeric protein is present in the amount of about 30 micrograms per dose.

8. The vaccine of any one of claims 1-7, wherein said CpG-containing immunostimulatory oligonucleotide a P-class immunostimulatory oligonucleotide characterized by the presence of one or more 5'-TLR-9 activating motif (s) and two palindromes or two complementarity areas.

9. The vaccine according to claim 8 wherein said P-class immunostimulatory oligonucleotide is 5' modified.

10. The vaccine according to claim 9, wherein said P class immunostimulatory oligonucleotide comprises at least 22 contiguous nucleotides of SEQ ID NO: 8.

11. The vaccine according to any one of claims 1-10, wherein the CpG containing immunostimulatory oligonucleotide is present in the amount of about 20 to about 250 μg per dose.

12. The vaccine according to any one of claims 1-10, wherein said metabolizable oil is plant- derived, animal-derived, or synthetic.

13. The vaccine according to claim 12, wherein said metabolizable oil is squalane.

14. The vaccine according to any one of claims 1-13, comprising said optional surfactant.

15. The vaccine according to claim 14, wherein the surfactant is polyoxyethylene sorbitan monooleate.

16. The vaccine of claim 15, whereina) said metabolizable oil is present in the amount of about 0.2% to about 0.8% v / v of the vaccine;b) said polyoxyethylene-polyoxypropylene block copolymer is present in the amount of about 0.1% to about 0.4% v / v of the vaccine; andc) said polyoxyethylene sorbitan monooleate is present in the amount of about 0.016% to about 0.064% v / v of the vaccine.

17. The vaccine according to claim 16, whereina) said metabolizable oil is present in the amount of about 0.4% v / v of the vaccine b) said polyoxyethylene-polyoxypropylene block copolymer is present in the amount of about 0.2% v / v of the vaccine; andc) said polyoxyethylene sorbitan monooleate is present in the amount of about 0.032% v / v of the vaccine.

18. A method of protecting an equine in need thereof against Lyme disease, the method comprising administering to said equine the vaccine according to any one of claims 1-17.

19. The method of claim 18, wherein the step of administering comprises a first administration of the vaccine according to any one of claims 1-15 and a second administration of the vaccine according to any one of claims 1-15, wherein said first administration precedes said second administration by about 14 to about 42 days.

20. The method according to claim 18, further comprising a third administration of the vaccine according to any one of claims 1-15, wherein said third administration follows the second administration by about 14 to about 42 days.

21. The method according to claim 19 or 20, wherein said first administration precedes said second administration by about 21 days.

22. The method according to any one of claims 19-21, wherein said third administration follows said second administration by about 21 days.

23. The method according to any one of claims 16-18, wherein said equine is about 3 months of age or older.

24. The method according to any one of claims 16-18, wherein said equine is about 6 months of age or older.

25. The method according to any one of claims 18-24, wherein said equine has not been previously vaccinated against Lyme disease.

26. The method of claims 18-25, wherein said OspA antigen is present in the amount of about 5 micrograms per dose and wherein said chimeric protein is present in the amount of about 7.5 micrograms per dose.

27. The method according to claim 26, wherein said equine has not been previously vaccinated against Lyme disease.

28. The method according to claim 18 or 23, further comprising re-vaccination of said equine 18 months after the previous dose of the vaccine according to any one of claims 1-17.

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